PROJECT SUMMARY Myalgic encephalomyelitis, also known as chronic fatigue syndrome (ME/CFS), is a complex, debilitating disease that has baffled researchers for decades. Its inaccurate yet frequent dismissal as a psychosomatic condition and lack of recognition by many in the biomedical community have greatly hindered research; as a result, very little is known about its cause(s), and no biological diagnosis or approved treatments are available. Recent developments have more clearly defined this mysterious illness, and it is now clear that it afflicts up to 2.5 million in the United States and millions more worldwide. While the symptoms present at various levels, including neurological and cognitive, widespread molecular and immunological abnormalities have also been observed. This is consistent with a majority of patients reporting infection prior to the onset of ME/CFS, although it remains unclear why common infections would serve as triggers for a chronic illness only in some people. Nevertheless, there is compelling evidence for an active immune response in ME/CFS, as suggested by elevated levels of signalling molecules called cytokines and activity of killer T cells, which are triggered in cases of infection or autoimmunity. This proposal aims to uncover the immunological basis of ME/CFS, by characterizing T cell activity and genetic factors that may be contributing to it using cutting-edge technologies invented by this team. Firstly, the activity of different T cells will be examined using single-cell DNA sequencing methods that will determine the extent and nature of their activation, and its regulation by gene expression. Secondly, the human leukocyte antigen (HLA) locus ? the most challenging region of the human genome to sequence, and the most relevant to individual differences in immunology ? will be sequenced in a large cohort of ME/CFS patients to determine whether HLA variants may be contributing to the T cell activity observed, and/or to increased susceptibility to the disease. Finally, the molecular triggers of the immune response will be hunted using cell-free DNA sequencing to detect pathogens, and through methods to identify which molecules are being targeted by the activated T cells. Taken together, these findings will help to identify the molecular and immunological factors that trigger and/or sustain ME/CFS as a chronic illness, and whether its basis is infectious, autoimmune, or both. More broadly, this project will build a precise framework for ME/CFS as a molecular and immunological disease, opening up broad new possibilities for research, diagnosis, and treatment. Understanding the molecular nature of the immune response in ME/CFS may lead to the definition of clinically valuable subtypes, refined diagnostics, risk prediction, and personalized immunomodulatory therapies. Moreover, the similarity of ME/CFS to other medically challenging diseases like Lyme disease, multiple sclerosis, Gulf War Illness, fibromyalgia, and more means that the insights derived here could be relevant to many millions of patients.